EP0242833A1 - Process for the manufacture of L-amino acids by transamination - Google Patents

Process for the manufacture of L-amino acids by transamination Download PDF

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Publication number
EP0242833A1
EP0242833A1 EP87105790A EP87105790A EP0242833A1 EP 0242833 A1 EP0242833 A1 EP 0242833A1 EP 87105790 A EP87105790 A EP 87105790A EP 87105790 A EP87105790 A EP 87105790A EP 0242833 A1 EP0242833 A1 EP 0242833A1
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Prior art keywords
acid
transamination
aspartic acid
amino acids
mmol
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French (fr)
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EP0242833B1 (en
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Johann Dr. Then
Hans-Matthias Dr. Deger
Gerhard Dr. Wöhner
Hartmut Dr. Voelskow
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Hoechst AG
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Hoechst AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • Amino acids have a diverse application profile. They are used as a supplement in animal nutrition, as a food additive for humans or as part of infusion solutions. L-phenylalanine is also used as a synthetic building block for the sweetener aspartame, which consists of phenylalanine methyl ester and aspartic acid.
  • European patent application 152 275 describes in particular a process for the production of phenylalanine by transamination with the aid of a genetically modified microorganism which is characterized by overproduction of the aminotransferase.
  • the transamination reaction is carried out at at least 40 ° C., since at these higher temperatures the microorganism cells more permeabilize.
  • L-amino acids are prepared in such a way that ⁇ -keto acids are reacted with L-aspartic acid in the presence of a transaminase which has been isolated from E. coli.
  • the ⁇ -amino acid corresponding to the keto acid and oxaloacetate are formed from the aspartic acid.
  • the invention thus relates to a process for the microbial production of L-amino acids by transamination of an ⁇ -keto acid with the aid of an amino group donor, which is characterized in that aspartic acid, aspartic acid, aspartic acid, glutamine and glutamic acid are used and the reaction solution is gassed.
  • microorganisms are able to convert ⁇ -keto acids into L-amino acids by biotransformation. These microorganisms can be used according to the invention. However, Paracoccus denitrificans DSM 65 and streptomycetes isolated from soil samples are preferred. Best results are achieved with E. coli ATCC 11303. It is advantageous, but not absolutely necessary, that selection and mutation, in a manner known per se, in particular according to the process of German patent application DE 3 423 936, select microorganisms for increasing work against increasing amounts of the corresponding ⁇ -keto acid in the cultivation media which, due to their adaptation to the ⁇ -keto acid, carry out the biotransformation in better yields.
  • transaminase activities of 100-200 ⁇ mol / min ⁇ l culture fluid can be used.
  • up to 60 g / l of ⁇ -keto acid can be transaminated with the process according to the invention to the corresponding amino acid in about 100% yield.
  • the microorganisms are advantageously grown in a nutrient medium that is optimal for their growth under appropriate favorable temperature and ventilation conditions up to a wet weight of about 4 to 10 g / l nutrient solution.
  • the conditions which are most favorable for the particular microorganism are either known to the person skilled in the art or can be determined in simple preliminary tests.
  • the cells are then used in the nutrient solution or separated from the nutrient solution for the amination of the keto acids.
  • the transamination can be carried out with whole or with disrupted cells, the usual disruption methods being used. To make work easier, intact cells are preferred. It is also possible to use the microorganisms in a fixed form.
  • the known methods are considered for fixation, advantageously the methods according to German Offenlegungsschriften 32 37 341 (US 4 603 111) and 32 43 591 (US 4 542 069).
  • the microorganisms are suspended in a buffer physiological for the cells with the addition of the ⁇ -keto acid and the amino group donor.
  • the enzymatic activity added to the batch can fluctuate over a wide range. It is expediently between 10 and 20,000 ⁇ mol / min ⁇ l.
  • the batch preferably contains cell quantities with an enzyme activity of 1500-2000 ⁇ mol / min ⁇ l.
  • Amino acids such as glycine alanine, valine, leucine, are used as amino group donors, in particular asparagine, aspartic acid, glutamine and glutamic acid. These amino acids are used in the form of their free acid or suitable salts (depending on the medium used).
  • the amino group donor is used in equimolar amounts or in excess of the ⁇ -keto acid. Ratios of 1: 1 to 5: 1, advantageously 1: 1 to 2: 1, have proven successful.
  • the reaction components can be added to the reaction mixture as a solution in water or by adding the solid substances simultaneously.
  • a staggered or continuous addition in amounts of 1-4.5%, in particular 1.5-2%, in each case based on the weight of the reaction mixture over a period of 1-90 hours, preferably 2-40 hours, is preferred.
  • the cheapest procedure depends on the particular microorganism and can easily be determined in simple preliminary tests.
  • Compressed air and pure nitrogen are preferred due to the low price and their accessibility.
  • ⁇ -keto acids can be aminated with the process according to the invention.
  • Amino acids which are incorporated into natural proteins, in particular those which are listed in the table, are preferably used.
  • Escherichia coli ATTC 11303 was cultivated according to conventional methods and mutagenized with N-methyl-N-nitro-N-nitroguanidine (MNG).
  • MNG N-methyl-N-nitro-N-nitroguanidine
  • the cells treated with MNG were spread on an autoclaved agar with the following composition: pH 7.2 was adjusted with sodium hydroxide solution.
  • a sterile-filtered solution of phenyl pyruvate was poured into the still hot agar, so that a final concentration of 24 g / l phenyl pyruvate was reached.
  • the plates were incubated at 37 ° C for 4 days. Colonies with a diameter of> 1 mm were isolated. 20% of the growing strains had increased transaminase activity compared to the parent strain.
  • the transaminase activity determination was carried out with the Sigma test kit G 0390. Instead of ⁇ -ketoglutarate, 12 mmol / l phenylpyruvate sodium salt was used.
  • the mutant of Escherichia coli ATCC 11303 selected according to Example 1 was cultivated in the nutrient solution from Example 1 without agar. It had a transaminase activity of 170 ⁇ mol / min ⁇ l after 20 hours of growth at 37 ° C.
  • the cells were centrifuged off, washed with 50 mmol / l phosphate buffer pH 7.4 and suspended in 30 mmolar phosphate buffer (pH 7.4) so that the suspension contained 1500 ⁇ mol / min ⁇ l transaminase activity. 24 g / l Na phenyl pyruvate and 20 g / l aspartic acid were added to the cell suspension.
  • the reaction mixture contained 21 g / l L-phenylalanine.
  • the solution was filtered and the water evaporated 1:10 concentrated. Phenylalanine was crystallized at pH 5.5 and 5 ° C.
  • the qualitative and quantitative determination of the amino acid was carried out by HPLC on an RP 18 column.
  • Cell material obtained according to Example 1 was suspended in 100 ml of a solution of 42 g / l phenylpyruvate, 34 g / l aspartic acid and 10 mmol / l phosphate buffer pH 7.4, so that the enzyme activity in the solution corresponds to 1,500 ⁇ mol / l ⁇ min .
  • Half of the reaction mixture was stirred at 37 ° C., while the other half was additionally gassed with 1 l of compressed air per liter of reaction buffer and minute. After 4 hours, the non-aerated reaction vessel contained 16.5 g / l phenylalanine, while 26.9 g / l were measured in the aerated vessel.
  • Cells from Paracoccus denitrificans DSM 65 were suspended in 100 ml of an aqueous solution of the following composition: 90 mmol / l aspartic acid, 27 ⁇ mol / l N-cetyl-N, N, N -trimethyl-ammonium bromide, 20 mmol / l 4-hydroxyphenylpyruvate and 30 mmol / l phosphate buffer (pH 7.4).
  • Cell material as in Example 3 was dissolved in 100 ml of an aqueous solution of 90 mmol / l aspartic acid, 27 ⁇ mol / l N-cetyl-N, N, N-trimethylammonium bromide, 35 mmol / l dimethylpyruvate and 30 mmol / l phosphate buffer (pH 7, 4) incubated. After 20 hours at 40 ° C and a gassing of 1 VVm with compressed air, 30 mmol / l valine could be measured.
  • Cell material as in Example 3 was incubated in 100 ml of an aqueous solution of 320 mmol / l phenylpyruvate sodium salt, 340 mmol / l aspartic acid, 10 ⁇ mol / l toluene and 10 mmol / l Tris / HCl buffer (pH 7.4). After 5 hours with a gassing of 0.5 VVm with oxygen, a phenylalanine content of 150 mmol / l could be measured at 37 ° C and a phenylalanine content of 270 mmol / l at 50 ° C.
  • Cell material as in Example 3 was suspended in 100 ml of an aqueous solution of 30 g / l phenylpyruvate sodium salt, 28 g / l aspartic acid and 10 mmol / l Tris / HCl buffer (pH 7.4).
  • the suspension was incubated at 50 ° C with gassing with 0.5 VVm compressed air. After 2 hours a phenylalanine concentration of 25 g / l could be measured.
  • 30 g of solid phenylpyruvate sodium salt and 26 g of solid aspartic acid sodium salt were then metered in per liter of reaction volume. After a further 2 hours, a phenylalanine concentration of 45 g / l was measured.

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Abstract

The yield of L-amino acids by transamination of alpha -keto acids using microorganisms can be substantially increased by gassing.

Description

Aminosäuren haben ein vielfältiges Anwendungsprofil. Sie finden ihren Einsatz als Supplement in der Tierernährung, als Nahrungsmitteladditiv für den Menschen oder als Be­standteil von Infusionslösungen. L-Phenylalanin findet eine weitere Anwendung als Synthesebaustein für den Süß­stoff Aspartam, der aus Phenylalaninmethylester und Aspa­raginsäure besteht.Amino acids have a diverse application profile. They are used as a supplement in animal nutrition, as a food additive for humans or as part of infusion solutions. L-phenylalanine is also used as a synthetic building block for the sweetener aspartame, which consists of phenylalanine methyl ester and aspartic acid.

Die Herstellung von L-Aminosäuren durch Biotransformation mit Transaminasen ist an sich bekannt. In der Europäischen Patentanmeldung 152 275 wird insbesondere ein Verfahren zur Herstellung von Phenylalanin durch Transaminierung mit Hilfe eines genetisch modifizierten Mikroorganismus be­schrieben, der sich durch Überproduktion der Aminotransfe­rase auszeichnet. Die Transaminierungsreaktion wird bei mindestens 40 °C durchgeführt, da bei diesen höheren Tem­peraturen die Mikroorganismenzellen stärker permeabilisie­ren.The production of L-amino acids by biotransformation with transaminases is known per se. European patent application 152 275 describes in particular a process for the production of phenylalanine by transamination with the aid of a genetically modified microorganism which is characterized by overproduction of the aminotransferase. The transamination reaction is carried out at at least 40 ° C., since at these higher temperatures the microorganism cells more permeabilize.

Nach der Europäischen Patentanmeldung 135 846 (US 4 518 692; US 45 25 454) erfolgt die Herstellung von L-Aminosäuren derart, daß α-Ketosäuren mit L-Asparaginsäure in Gegenwart einer Transaminase umgesetzt werden, die aus E. coli iso­liert wurde. Es entstehen die der Ketosäure entsprechende α-Aminosäure sowie Oxalacetat aus der Asparaginsäure. Durch Entfernung von Oxalacetat aus dem Reaktionsmedium nach der Decarboxylierung, wird das Reaktionsgleichgewicht auf die Seite des Endprodukts verschoben. Oxalacetat decar­boxyliert in wäßriger Lösung aufgrund seiner Instabilität. Diese Reaktion kann thermisch, chemisch oder enzymatisch beschleunigt werden.According to European patent application 135 846 (US 4,518,692; US 4,525,454), L-amino acids are prepared in such a way that α-keto acids are reacted with L-aspartic acid in the presence of a transaminase which has been isolated from E. coli. The α-amino acid corresponding to the keto acid and oxaloacetate are formed from the aspartic acid. By removing oxaloacetate from the reaction medium after decarboxylation, the reaction equilibrium is shifted to the end product side. Oxaloacetate decarboxylates in aqueous solution due to its instability. This reaction can be accelerated thermally, chemically or enzymatically.

Die Selektion und Mutation von Mikroorganismen aus der Reihe E coli., Paracoccus denitrificans, Torula, Rhodoto­rula und Streptomyces zur Herstellung von L-Phenylalanin aus Phenylbrenztraubensäure mit verbesserter Ausbeute wird in der Deutschen Patentanmeldung DE 3 423 936 dargestellt.The selection and mutation of microorganisms from the series E coli., Paracoccus denitrificans, Torula, Rhodotorula and Streptomyces for the production of L-phenylalanine from phenylpyruvic acid with improved yield is shown in German patent application DE 3 423 936.

Überraschend wurde nun gefunden, daß durch einfaches Be­gasen der Reaktionslösung, in der die Transaminierung durchgeführt wird, nach kurzer Reaktionszeit eine Ausbeute der gewünschten Aminosäure von bis zu 100 % erzielt werden kann.Surprisingly, it has now been found that by simply gassing the reaction solution in which the transamination is carried out, a yield of the desired amino acid of up to 100% can be achieved after a short reaction time.

Die Erfindung betrifft somit ein Verfahren zur mikrobiel­len Herstellung von L-Aminosäuren durch Transaminierung ei­ner α-Ketosäure mit Hilfe eines Aminogruppendonators, das dadurch gekennzeichnet ist, daß als Aminogruppendonator Asparagin, Asparaginsäure, Glutamin und Glutaminsäure einge setzt werden und die Reaktionslösung begast wird.The invention thus relates to a process for the microbial production of L-amino acids by transamination of an α-keto acid with the aid of an amino group donor, which is characterized in that aspartic acid, aspartic acid, aspartic acid, glutamine and glutamic acid are used and the reaction solution is gassed.

Im folgenden wird die Erfindung genau erläutert bzw. in den Patentansprüchen definiert.The invention is explained in detail below or defined in the patent claims.

Zahlreiche Mikroorganismen sind in der Lage, α-Ketosäuren durch Biotransformation in L-Aminosäure umzuwandeln. Diese Mikroorganismen können erfindungsgemäß einge­setzt werden. Bevorzugt wird jedoch mit Paracoccus deni­trificans DSM 65 sowie mit aus Erdbodenproben isolierten Streptomyceten gearbeitet. Beste Ergebnisse werden mit E. coli ATCC 11303 erzielt. Es ist vorteilhaft, jedoch nicht unbedingt notwendig, daß durch Selektion und Muta­tion, in an sich bekannter Weise, insbesondere gemäß dem Verfahren der Deutschen Patentanmeldung DE 3 423 936, gegen steigende Mengen der entsprechenden α-Ketosäure in den Anzuchtmeden für die weiteren Arbeiten Mikroorganismen ausgewählt werden, die aufgrund ihrer Adaptation an die α-Ketosäure die Biotransformation in besseren Ausbeuten durchführen.Numerous microorganisms are able to convert α-keto acids into L-amino acids by biotransformation. These microorganisms can be used according to the invention. However, Paracoccus denitrificans DSM 65 and streptomycetes isolated from soil samples are preferred. Best results are achieved with E. coli ATCC 11303. It is advantageous, but not absolutely necessary, that selection and mutation, in a manner known per se, in particular according to the process of German patent application DE 3 423 936, select microorganisms for increasing work against increasing amounts of the corresponding α-keto acid in the cultivation media which, due to their adaptation to the α-keto acid, carry out the biotransformation in better yields.

Durch Selektion können bespielsweise Transaminaseaktivi­täten von 100-200 µmol/min ·l Kulturflüssigkeit zugrunde gelegt werden. Auf diese Weise können mit dem erfindungs­gemäßen Verfahren bis zu 60 g/l α-Ketosäure mit etwa 100 %iger Ausbeute zu der entsprechenden Aminosäure transami­niert werden.By selection, for example, transaminase activities of 100-200 µmol / min · l culture fluid can be used. In this way, up to 60 g / l of α-keto acid can be transaminated with the process according to the invention to the corresponding amino acid in about 100% yield.

Die Mikroorganismen werden vorteilhaft in einem für ihr Wachstum optimalen Nährmedium unter entsprechenden günsti­gen Temperatur- und Belüftungsbedingungen bis zu einem Feuchtgewicht von etwa 4 bis 10 g/l Nährlösung angezogen. Die für den jeweiligen Mikroorganismus günstigsten Bedin­gungen sind dem Fachmann entweder bekannt oder können in einfachen Vorversuchen festgestellt werden. Die Zellen werden dann in der Nährlösung oder von der Nährlösung ab­getrennt zur Aminierung der Ketosäuren eingesetzt. Die Transaminierung kann mit ganzen oder auch mit aufgeschlos­senen Zellen durchgeführt werden, wobei die üblichen Auf­schlußmethoden angewendet werden. Aus Gründen der Arbeits­erleichterung wird bevorzugt mit intakten Zellen gearbei­tet. Es ist weiterhin möglich, die Mikroorganismen in fi­xierter Form einzusetzen. Für die Fixierung kommen die be­kannten Verfahren in Betracht, vorteilhaft die Methoden nach den Deutschen Offenlegungsschriften 32 37 341 (US 4 603 111) und 32 43 591 (US 4 542 069).The microorganisms are advantageously grown in a nutrient medium that is optimal for their growth under appropriate favorable temperature and ventilation conditions up to a wet weight of about 4 to 10 g / l nutrient solution. The conditions which are most favorable for the particular microorganism are either known to the person skilled in the art or can be determined in simple preliminary tests. The cells are then used in the nutrient solution or separated from the nutrient solution for the amination of the keto acids. The transamination can be carried out with whole or with disrupted cells, the usual disruption methods being used. To make work easier, intact cells are preferred. It is also possible to use the microorganisms in a fixed form. The known methods are considered for fixation, advantageously the methods according to German Offenlegungsschriften 32 37 341 (US 4 603 111) and 32 43 591 (US 4 542 069).

Die Mikroorganismen werden in einem für die Zellen phy­siologischen Puffer suspendiert unter Zugabe der α-Keto­säure und des Aminogruppendonators. Je nach Menge der Mikroorganismen kann die dem Ansatz zugegebene enzymatische Aktivität in weiten Bereichen schwanken. Zweckmäßig liegt sie zwischen 10 bis 20.000 µmol/min·l. Vorzugsweise ent­hält der Ansatz Zellmengen mit einer Enzymaktivität von 1500-2000 µmol/min·l.The microorganisms are suspended in a buffer physiological for the cells with the addition of the α-keto acid and the amino group donor. Depending on the amount of microorganisms, the enzymatic activity added to the batch can fluctuate over a wide range. It is expediently between 10 and 20,000 µmol / min · l. The batch preferably contains cell quantities with an enzyme activity of 1500-2000 μmol / min · l.

Als Aminogruppendonator finden Aminosäuren, wie z.B. Glycin Alanin, Valin, Leucin, Anwendung, insbeondere Asparagin, Aspa­raginsäure, Glutamin und Glutaminsäure. Diese Aminosäuren werden in Form ihrer freien Säure oder geeigneten Salze (entsprechend dem verwendeten Medium) eingesetzt. Der Ami­nogruppendonator wird in äquimolaren Mengen bzw. im Über­schuß zur α-Ketosäure eingesetzt. Verhältnisse von 1:1 bis 5:1, vorteilhaft 1:1 bis 2:1, haben sich bewährt.Amino acids, such as glycine alanine, valine, leucine, are used as amino group donors, in particular asparagine, aspartic acid, glutamine and glutamic acid. These amino acids are used in the form of their free acid or suitable salts (depending on the medium used). The amino group donor is used in equimolar amounts or in excess of the α-keto acid. Ratios of 1: 1 to 5: 1, advantageously 1: 1 to 2: 1, have proven successful.

Die zugabe der Reaktionskomponenten zum Reaktionsansatz kann als Lösung in Wasser oder durch Zugabe der festen Substanzen gleichzeitig erfolgen. Bevorzugt ist jedoch eine gestaffelte bzw. kontinuierliche Zugabe in Mengen von 1-4,5 %, insbesondere 1,5-2 %, jeweils bezogen auf das Gewicht des Reaktions­ansatzes über einen Zeitraum von 1-90 Stunden, vorzugs­weise 2-40 Stunden.The reaction components can be added to the reaction mixture as a solution in water or by adding the solid substances simultaneously. However, a staggered or continuous addition in amounts of 1-4.5%, in particular 1.5-2%, in each case based on the weight of the reaction mixture over a period of 1-90 hours, preferably 2-40 hours, is preferred.

Es wird vorteilhaft bei einem pH-Wert zwischen 5 und 9 insbesondere zwischen 7 und 8,5, gearbeitet. Es ist außer­dem zweckmäßig, die Transaminierung in einem Temperatur­bereich von 20°-65°C durchzuführen. Bei niedrigeren Tem­peraturen verläuft die Enzym-Reaktion zunehmend langsamer, während das Enzym bei höheren Temperaturen fortschreitend desaktiviert wird.It is advantageous to work at a pH between 5 and 9, in particular between 7 and 8.5. It is also advisable to carry out the transamination in a temperature range of 20 ° -65 ° C. The enzyme reaction progressively slows down at lower temperatures, while the enzyme progressively deactivates at higher temperatures.

Die günstigste Vorgehensweise hängt von dem jeweiligen Mi­kroorganismus ab und kann in einfachen Vorversuchen leicht festgelegt werden.The cheapest procedure depends on the particular microorganism and can easily be determined in simple preliminary tests.

Es hat sich besonders bewährt, die Mikroorganismen vor bzw. während der Transaminierungsreaktion zu permeabilisieren. Dies kann durch Zugabe geeigneter Agenzien, wie Toluol, Cetyltrimethylammoniumbromid, Dimethylsulfoxid etc., zum Inkubationsmedium erfolgen.It has proven particularly useful to permeabilize the microorganisms before or during the transamination reaction. This can be done by adding suitable agents, such as toluene, cetyltrimethylammonium bromide, dimethyl sulfoxide etc., to the incubation medium.

Hohe Umsatzraten in kurzer Zeit werden überraschenderweise dann erreicht, wenn die beschriebenen Reaktionsansätze, in denen die Biotransformation stattfindet, begast werden. Die Begasung erfolgt im Bereich von 0.1-15 VVm, vorteil­haft im Bereich von 0,2-3 VVm. Es können grundsätzlich alle Gase eingesetzt werden, die die Enzymaktivität des Mi­kroorganismus nicht wesentlich heruntersetzen. Geeignet sind beispielsweise Preßluft, reiner Sauerstoff, Stick­stoff aber auch verschiedene Edelgase, wie Helium, Neon, Argon oder Krypton.High conversion rates in a short time are surprisingly achieved when the described reaction batches in which the biotransformation takes place are gassed. Fumigation takes place in the range of 0.1-15 VVm, advantageously in the range of 0.2-3 VVm. Basically it can all gases are used that do not significantly reduce the enzyme activity of the microorganism. For example, compressed air, pure oxygen, nitrogen but also various noble gases such as helium, neon, argon or krypton are suitable.

Aufgrund des günstigen Preises und ihrer Zugänglichkeit sind Preßluft und reiner Stickstoff bevorzugt.Compressed air and pure nitrogen are preferred due to the low price and their accessibility.

Mit dem erfindungsgemäßen Verfahren können grundsätzlich alle α-Ketosäuren aminiert werden. Bevorzugt werden Amino­säuren eingesetzt, die in natürliche Proteine eingebaut werden, insbesondere solche, die in der Tabelle aufgeführt sind.

Figure imgb0001
In principle, all α-keto acids can be aminated with the process according to the invention. Amino acids which are incorporated into natural proteins, in particular those which are listed in the table, are preferably used.
Figure imgb0001

Die anschließenden Beispiele dienen dazu, die vorgestellte Erfindung weiter zu illustrieren. Prozentangaben beziehen sich, wenn nicht anders angegeben, auf das Gewicht.The following examples serve to further illustrate the presented invention. Unless otherwise stated, percentages relate to the weight.

Beispiel 1example 1

Escherichia coli ATTC 11303 wurde nach konventionellen Me­thoden kultiviert und mit N-Methyl-N-nitro-N-nitroguanidin (MNG) mutagenisiert. Die mit MNG behandelten Zellen wurden auf einen autoklavierten Agar mit folgender Zusammensetzung ausgestrichen:

Figure imgb0002
pH 7,2 wurde mit Natronlauge eingestellt.Escherichia coli ATTC 11303 was cultivated according to conventional methods and mutagenized with N-methyl-N-nitro-N-nitroguanidine (MNG). The cells treated with MNG were spread on an autoclaved agar with the following composition:
Figure imgb0002
 pH 7.2 was adjusted with sodium hydroxide solution.

Eine sterilfiltrierte Lösung von Phenylpyruvat wurde in den noch heißen Agar eingegossen, so daß eine Endkonzen­tration von 24 g/l Phenylpyruvat erreicht wurde. Die Plat­ten wurden bei 37 °C 4 Tage inkubiert. Kolonien mit einem Durchmesser von >1 mm wurden isoliert. 20 % der wachsenden Stämme hatten eine erhöhte Transaminaseaktivität im Ver­gleich zum Ausgangsstamm.A sterile-filtered solution of phenyl pyruvate was poured into the still hot agar, so that a final concentration of 24 g / l phenyl pyruvate was reached. The plates were incubated at 37 ° C for 4 days. Colonies with a diameter of> 1 mm were isolated. 20% of the growing strains had increased transaminase activity compared to the parent strain.

Die Transaminaseaktivitätsbestimmung wurde mit dem Sigma Test-kit G 0390 durchgeführt. Statt α-Ketoglutarat wurde 12 mmol/l Phenylpyruvat-Natriumsalz eingesetzt.The transaminase activity determination was carried out with the Sigma test kit G 0390. Instead of α-ketoglutarate, 12 mmol / l phenylpyruvate sodium salt was used.

Beispiel 2Example 2

Die gemäß Beispiel 1 selektionierte Mutante von Escheri­chia coli ATCC 11303 wurde in der Nährlösung aus Beispiel 1 ohne Agar kultiviert. Sie hatte eine Transaminaseaktivi­tät von 170 µmol/min·l nach 20 Stunden Wachstum bei 37 °C. Die Zellen wurden abzentrifugiert, mit 50 mmol/l Phosphat­puffer pH 7,4 gewaschen und in 30 mmolarem Phosphatpuffer (pH 7,4) suspendiert, so daß die Suspension 1500 µmol/min·l Transaminaseaktivität enthielt. 24 g/l Na-Phenylpyruvat und 20 g/l Asparaginsäure wurden in die Zellsuspension gegeben.The mutant of Escherichia coli ATCC 11303 selected according to Example 1 was cultivated in the nutrient solution from Example 1 without agar. It had a transaminase activity of 170 µmol / min · l after 20 hours of growth at 37 ° C. The cells were centrifuged off, washed with 50 mmol / l phosphate buffer pH 7.4 and suspended in 30 mmolar phosphate buffer (pH 7.4) so that the suspension contained 1500 μmol / min · l transaminase activity. 24 g / l Na phenyl pyruvate and 20 g / l aspartic acid were added to the cell suspension.

Nach einer Inkubationszeit von 6 Stunden bei 37 °C ent­hielt das Reaktionsgemisch 21 g/l L-Phenylalanin. Die Lö­sung wurde filtriert und durch Verdampfen des Wassers auf 1:10 konzentriert. Phenylalanin wurde bei pH 5,5 und 5 °C kristallisiert. Die qualitative und quantitative Bestim­mung der Aminosäure erfolgte durch HPLC auf einer RP 18-­Säule.After an incubation period of 6 hours at 37 ° C., the reaction mixture contained 21 g / l L-phenylalanine. The solution was filtered and the water evaporated 1:10 concentrated. Phenylalanine was crystallized at pH 5.5 and 5 ° C. The qualitative and quantitative determination of the amino acid was carried out by HPLC on an RP 18 column.

Beispiel 3Example 3

Gemäß Beispiel 1 erhaltenes Zellmaterial wurde in 100 ml einer Lösung aus 42 g/l Phenylpyruvat, 34 g/l Asparagin­säure und 10 mmol/l Phosphatpuffer pH 7,4 suspendiert, so daß die Enzymaktivität in der Lösung 1 500 µmol/l·min ent­spricht. Eine Hälfte des Reaktionsansatzes wurde bei 37 °C gerührt, während die andere Hälfte noch zusätzlich mit 1 l Preßluft pro Liter Reaktionspuffer und Minute begast wurde. Nach 4 Stunden enthielt das nicht belüftete Reak­tionsgefäß 16,5 g/l Phenylalanin, während im belüfteten Ge­fäß 26,9 g/l gemessen wurden.Cell material obtained according to Example 1 was suspended in 100 ml of a solution of 42 g / l phenylpyruvate, 34 g / l aspartic acid and 10 mmol / l phosphate buffer pH 7.4, so that the enzyme activity in the solution corresponds to 1,500 µmol / l · min . Half of the reaction mixture was stirred at 37 ° C., while the other half was additionally gassed with 1 l of compressed air per liter of reaction buffer and minute. After 4 hours, the non-aerated reaction vessel contained 16.5 g / l phenylalanine, while 26.9 g / l were measured in the aerated vessel.

Beispiel 4Example 4

Zellen von Paracoccus denitrificans DSM 65, deren Menge einer Enzymaktivität von 100 µmol/l·min entspricht, wurde in 100 ml einer wäßrigen Lösung folgender Zusammensetzung suspen­diert: 90 mmol/l Asparaginsäure, 27 µmol/l N-Cetyl-N,N,N-­trimethyl-ammoniumbromid, 20 mmol/l 4-Hydroxyphenylpyruvat und 30 mmol/l Phosphatpuffer (pH 7,4).Cells from Paracoccus denitrificans DSM 65, the amount of which corresponds to an enzyme activity of 100 μmol / l · min, were suspended in 100 ml of an aqueous solution of the following composition: 90 mmol / l aspartic acid, 27 μmol / l N-cetyl-N, N, N -trimethyl-ammonium bromide, 20 mmol / l 4-hydroxyphenylpyruvate and 30 mmol / l phosphate buffer (pH 7.4).

Nach 20 Stunden Inkubation bei 30 °C und einer Begasung von 0,5 VVm mit Stickstoff konnten 18 mmol/l L-Tyrosin ge­messen werden.After 20 hours of incubation at 30 ° C and a gassing of 0.5 VVm with nitrogen, 18 mmol / l L-tyrosine could be measured.

Beispiel 5Example 5

Zellmaterial wie in Beispiel 3 wurde in 100 ml einer wäß­rigen Lösung aus 90 mmol/l Asparaginsäure, 27 µmol/l N-Ce­tyl-N,N,N-trimethylammoniumbromid, 35 mmol/l Dimethylpyru­vat und 30 mmol/l Phosphatpuffer (pH 7,4) inkubiert. Nach 20 Stunden bei 40 °C und einer Begasung von 1 VVm mit Preß­luft konnten 30 mmol/l Valin gemessen werden.Cell material as in Example 3 was dissolved in 100 ml of an aqueous solution of 90 mmol / l aspartic acid, 27 µmol / l N-cetyl-N, N, N-trimethylammonium bromide, 35 mmol / l dimethylpyruvate and 30 mmol / l phosphate buffer (pH 7, 4) incubated. After 20 hours at 40 ° C and a gassing of 1 VVm with compressed air, 30 mmol / l valine could be measured.

Beispiel 6Example 6

Zellmaterial wie in Beispiel 3 wurde in 100 ml einer wäß­rigen Lösung aus 320 mmol/l Phenylpyruvat Natriumsalz, 340 mmol/l Asparaginsäure, 10 µmol/l Toluol und 10 mmol/l Tris/HCl-Puffer (pH 7,4) inkubiert. Nach 5 Stunden bei ei­ner Begasung von 0,5 VVm mit Sauerstoff konnten bei 37 °C ein Phenylalaningehalt von 150 mmol/l und bei 50 °C ein Phenylalaningehalt von 270 mmol/l gemessen werden.Cell material as in Example 3 was incubated in 100 ml of an aqueous solution of 320 mmol / l phenylpyruvate sodium salt, 340 mmol / l aspartic acid, 10 µmol / l toluene and 10 mmol / l Tris / HCl buffer (pH 7.4). After 5 hours with a gassing of 0.5 VVm with oxygen, a phenylalanine content of 150 mmol / l could be measured at 37 ° C and a phenylalanine content of 270 mmol / l at 50 ° C.

Beispiel 7Example 7

Zellmaterial wie in Beispiel 3 wurde in 100 ml einer wäß rigen Lösung aus 30 g/l Phenylpyruvat Natriumsalz, 28 g/l Asparaginsäure und 10 mmol/l Tris/HCl-Puffer (pH 7,4) sus­pendiert. Die Suspension wurde bei 50 °C unter Begasung mit 0,5 VVm Preßluft inkubiert. Nach 2 Stunden konnte eine Phenylalaninkonzentration von 25 g/l gemessen werden. Nun wurden 30 g festes Phenylpyruvat Natriumsalz und 26 g fe­ste Asparaginsäure Natriumsalz pro Liter Reaktionsvolumen zudosiert. Nach weiteren 2 Stunden konnte eine Phenylala­ninkonzentration von 45 g/l gemessen werden.Cell material as in Example 3 was suspended in 100 ml of an aqueous solution of 30 g / l phenylpyruvate sodium salt, 28 g / l aspartic acid and 10 mmol / l Tris / HCl buffer (pH 7.4). The suspension was incubated at 50 ° C with gassing with 0.5 VVm compressed air. After 2 hours a phenylalanine concentration of 25 g / l could be measured. 30 g of solid phenylpyruvate sodium salt and 26 g of solid aspartic acid sodium salt were then metered in per liter of reaction volume. After a further 2 hours, a phenylalanine concentration of 45 g / l was measured.

Claims (7)

1. Verfahren zur mikrobiellen Herstellung von L-Amino­säuren durch Transaminierung einer α-Ketosäure mit Hilfe eines Aminogruppendonators in wäßriger Lösung, dadurch gekennzeichnet, daß als Aminogruppendonator Asparagin, Asparaginsäure, Glutamin und Glutaminsäure eingesetzt werden und die Reaktionslösung begast wird.1. A process for the microbial production of L-amino acids by transamination of an α-keto acid with the aid of an amino group donor in aqueous solution, characterized in that aspartic acid, aspartic acid, aspartic acid, glutamine and glutamic acid are used and the reaction solution is gassed. 2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Reaktionslösung mit 0,1-15 VVm begast wird.2. The method according to claim 1, characterized in that the reaction solution is gassed with 0.1-15 VVm. 3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß die Reaktionslösung mit 0,2-3 VVm begast wird.3. The method according to claim 2, characterized in that the reaction solution is gassed with 0.2-3 VVm. 4. Verfahren nach einem oder mehreren der Ansprüche, 1 bis 3, dadurch gekennzeichnet, daß die Zugabe der α-Keto­säure und des Aminogruppendonators zum Reaktiosmedium gleichzeitig gestaffelt oder kontinuierlich über einen Zeitraum von 1-90 Stunden erfolgt.4. The method according to one or more of claims 1 to 3, characterized in that the addition of the α-keto acid and the amino group donor to the reaction medium simultaneously or staggered over a period of 1-90 hours. 5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, daß die Zugabe über 2-40 Stunden erfolgt.5. The method according to claim 4, characterized in that the addition takes place over 2-40 hours. 6. Verfahren nach einem oder mehreren der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß Aminogruppendonator und α-Ketosäure im Verhältnis 1:1 bis 5:1 (mol:mol) einge­setzt werden.6. The method according to one or more of claims 1 to 5, characterized in that amino group donor and α-keto acid in a ratio of 1: 1 to 5: 1 (mol: mol) are used. 7. Verfahren nach Anspruch 6 dadurch gekennzeichnet, daß Aminogruppendonator und α-Ketosäure im Verhältnis 1:1 bis 2:1 (mol:mol) eingesetzt werden.7. The method according to claim 6, characterized in that amino group donor and α-keto acid in a ratio of 1: 1 to 2: 1 (mol: mol) are used.
EP87105790A 1986-04-24 1987-04-18 Process for the manufacture of l-amino acids by transamination Expired - Lifetime EP0242833B1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0297619A2 (en) * 1987-07-02 1989-01-04 Hoechst Aktiengesellschaft Process for the preparation of L-phenyl alanine from benzylidene hydantoin

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0132999A2 (en) * 1983-07-29 1985-02-13 Rhone-Poulenc Chimie Process and compositions for preparing phenylalanine
DE3427495A1 (en) * 1983-08-05 1985-02-14 W.R. Grace & Co., New York, N.Y. METHOD FOR PRODUCING L-AMINO ACIDS FROM (ALPHA) KETO ACIDS
EP0135846A2 (en) * 1983-09-01 1985-04-03 Genetics Institute, Inc. Production of L-amino acids by transamination
FR2557887A1 (en) * 1984-01-05 1985-07-12 Grace W R Ltd PROCESS FOR PRODUCING L-PHENYLALANINE
BE902313A (en) * 1985-04-29 1985-08-16 Genex Corp Phenylalanine ammonia lyase enzyme prepn. - by cultivating microorganism, e.g. Rhodotorula, aerobically and then under static conditions anaerobically
EP0167058A2 (en) * 1984-06-29 1986-01-08 Hoechst Aktiengesellschaft Process for producing L-phenyl alanine
EP0152275B1 (en) * 1984-02-07 1991-06-19 THE NUTRASWEET COMPANY (a Delaware corporation) Production of amino acids via bioconversion

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0132999A2 (en) * 1983-07-29 1985-02-13 Rhone-Poulenc Chimie Process and compositions for preparing phenylalanine
DE3427495A1 (en) * 1983-08-05 1985-02-14 W.R. Grace & Co., New York, N.Y. METHOD FOR PRODUCING L-AMINO ACIDS FROM (ALPHA) KETO ACIDS
EP0135846A2 (en) * 1983-09-01 1985-04-03 Genetics Institute, Inc. Production of L-amino acids by transamination
FR2557887A1 (en) * 1984-01-05 1985-07-12 Grace W R Ltd PROCESS FOR PRODUCING L-PHENYLALANINE
EP0152275B1 (en) * 1984-02-07 1991-06-19 THE NUTRASWEET COMPANY (a Delaware corporation) Production of amino acids via bioconversion
EP0167058A2 (en) * 1984-06-29 1986-01-08 Hoechst Aktiengesellschaft Process for producing L-phenyl alanine
BE902313A (en) * 1985-04-29 1985-08-16 Genex Corp Phenylalanine ammonia lyase enzyme prepn. - by cultivating microorganism, e.g. Rhodotorula, aerobically and then under static conditions anaerobically

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Title
CHEMIKER ZEITUNG, Band 98, Nr. 11, November 1974, Seiten 523-532; G. NESEMANN et al.: "Technische Anwendung mikrobieller Verfahren" *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0297619A2 (en) * 1987-07-02 1989-01-04 Hoechst Aktiengesellschaft Process for the preparation of L-phenyl alanine from benzylidene hydantoin
EP0297619A3 (en) * 1987-07-02 1990-04-04 Hoechst Aktiengesellschaft Process for the preparation of l-phenyl alanine from benzylidene hydantoin

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PT84743A (en) 1987-05-01
JPS62257392A (en) 1987-11-09
NO871687D0 (en) 1987-04-23
DK206287D0 (en) 1987-04-23
DE3613952A1 (en) 1987-10-29
ZA872880B (en) 1987-11-25
EP0242833B1 (en) 1992-02-19
AU7191387A (en) 1987-10-29
NO871687L (en) 1987-10-26
PT84743B (en) 1989-06-08
IL82297A0 (en) 1987-10-30
DK206287A (en) 1987-10-25
FI871761A0 (en) 1987-04-22
FI871761A (en) 1987-10-25
CA1291925C (en) 1991-11-12
DE3776717D1 (en) 1992-03-26

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